Differential responses of chicken monocyte-derived dendritic cells infected with Salmonella Gallinarum and Salmonella Typhimurium

Salmonella enterica serovar Gallinarum is a host-restricted bacterial pathogen that causes a serious systemic disease exclusively in birds of all ages. Salmonella enterica serovar Typhimurium is a host-generalist serovar. Dendritic cells (DCs) are key antigen-presenting cells that play an important part in Salmonella host-restriction. We evaluated the differential response of chicken blood monocyte-derived dendritic cells (chMoDCs) exposed to S. Gallinarum or S. Typhimurium. S. Typhimurium was found to be more invasive while S. Gallinarum was more cytotoxic at the early phase of infection and later showed higher resistance against chMoDCs killing. S. Typhimurium promoted relatively higher upregulation of costimulatory and other immune function genes on chMoDCs in comparison to S. Gallinarum during early phase of infection (6 h) as analyzed by real-time PCR. Both Salmonella serovars strongly upregulated the proinflammatory transcripts, however, quantum was relatively narrower with S. Gallinarum. S. Typhimurium-infected chMoDCs promoted relatively higher proliferation of naïve T-cells in comparison to S. Gallinarum as assessed by mixed lymphocyte reaction. Our findings indicated that host restriction of S. Gallinarum to chicken is linked with its profound ability to interfere the DCs function. Present findings provide a valuable roadmap for future work aimed at improved vaccine strategies against this pathogen.


Isolation and culture of chicken blood monocyte-derived dendritic cells (chMoDCs). White
Leghorn broiler chicks (3 weeks old) were procured from the Indian Council of Agricultural Research-Central Avian Research Institute Hatchery (Izatnagar). They were screened for the presence of Salmonella species by the previously described method 13 .
Chicken DCs were prepared from buffy coats obtained from whole blood (collected via wing veins) in sterile vacutainers containing heparin and diluted with an equal volume of phosphate-buffered saline (PBS). Peripheral blood mononuclear cells (PBMCs) were isolated using the density-gradient method (Histopaque-1077; Sigma Life Sciences, Saint Louis, MO, USA). The cell pellet was suspended by addition of 500 μl of prewarmed RPMI-1640 medium (Gibco Life Technologies, Carlsbad, CA, USA). The concentration and viability of cells were determined using Trypan Blue (0.4%) staining. In vitro culture of chMoDCs was done according to the method described earlier with slight modifications 14 . PBMCs (2 × 10 6 cells/mL) were cultured in 24-well plates in RPMI-1640 complete medium containing 8% chicken serum, 2% fetal bovine serum (FBS), 1% non-essential amino acids, 1% l-glutamine, penicillin (1 U/mL) and streptomycin (1 μg/mL) at 37 °C in an atmosphere of 5% CO 2 for 6 days using recombinant chicken granulocyte-macrophage colony stimulating factor (GM-CSF; 20-50 ng/mL) (Abcam, USA) and interleukin (IL)-4 (10-30 ng/mL) (Abcam, USA). On every second day, threequarters of the medium was replaced with fresh, prewarmed complete RPMI-1640 medium containing GM-CSF and IL-4 to remove non-adherent cells. The growth and differentiation of cells was recorded by observing the morphology, cells aggregation, and growth pattern of cells every second day up to day-6 of culture. On day-6, chMoDCs were infected with S. Gallinarum and S. Typhimurium at a multiplicity of infection (MoI) of 10 for different intervals according to the needs of different assays. Cells treated with lipopolysaccharide (LPS; (1 μg/ mL) from Salmonella enterica serovar Typhimurium (Sigma, USA) and mock-treated cells were used as positive and negative controls, respectively. Morphologic changes in chMoDCs following infection with S. Gallinarum or S. Typhimurium were recorded ≤ 24 h post-infection. Intracellular survival assay (gentamicin protection assay). An assay to measure intracellular bacterial survival was undertaken according to the previously described method with slight modifications 15 . Following infection of chMoDCs with respective Salmonella serovars for 1.5 h, the medium was replaced with fresh RPMI-1640 containing gentamicin (50 μg/mL). Cells were incubated further at 37 °C in an atmosphere of 5% CO 2 for an additional 1.5 h to allow killing of extracellular bacteria. At 3 h after infection, cell lysates were prepared by addition of 0.1% Triton X-100 for bacterial counting, or the medium was replaced with fresh RPMI-1640 containing gentamicin (25 μg/mL) and re-incubation undertaken. Cell lysates were prepared at 24 h and 48 h after infection as described above, and counting of viable bacteria was done by plating serial dilutions , chemokines (C-X-C motif ligand 1 (CXCLi)1, CXCLi2) and toll-like receptor (TLR)-4 and TLR-21) from bacteria-infected and control groups was quantified using published primers (Supplementary Table S1). Beta-actin was employed as an endogenous reference gene to calculate ΔCt values for each target gene. Previously, this has been established that β-actin is a stable endogenous reference gene for RT-qPCR studies 17 . The individual sample was run in triplicate, each with a 20-μl reaction. Briefly, 10 ng of cDNA (1 μl) was mixed with 0.2 μl of each forward and reverse primers (10-pmol each) with 10 μl of 2 × SYBR Green Master Mix in a final volume of 20 μl with the following cycling conditions: one initial cycle at 95 °C for 5 min followed by 40 cycles of amplification with denaturation at 95 °C for 10 s, annealing at 47-64 °C for 30 s (for different genes) and extension at 72 °C for 30 s. The specificity of primers was confirmed by the amplification plot and dissociation curve. The 2 −ΔΔct method was employed to ascertain the relative expression of each target gene in Salmonella-infected chMoDcs as the fold-difference from the mock-infected control group (immature chMoDCs) 18 . The LPS-treated group (positive control) was used to access chMoDC maturation.
Mixed lymphocyte reaction (MLR) assay. The MLR assay was undertaken according to the method described by Cheminay and coworkers with slight modifications 19 . All reactions were set up in triplicate. Briefly, chMoDCs were cultured up to 6 days in 96-well plates as described above. On day-6, chMoDCs were infected with S. Gallinarum or S. Typhimurium at MoI = 10 in triplicate wells along with respective controls. After 1 h, non-internalized bacteria were removed by two washes with PBS. To kill the remaining extracellular bacteria, infected chMoDCs were incubated in medium containing gentamicin (100 μg/mL) for 1 h. After washing, chMoDCs were incubated further in the presence of medium containing gentamicin (25 μg/mL) for 24 h. The absence of extracellular bacteria was tested by plating supernatants onto HEA plates.
To carry out the MLR assay, T-lymphocytes were isolated and purified from allogenic chicken spleens (from chickens aged 3-6 weeks) using a nylon-wool column 20 . Purified T-cells were added to S. Gallinarum-or S. Typhimurium-primed chMoDCs (10:1) and incubated at 37 °C in an atmosphere of 5% CO 2 for 72 h. The tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well (20 μL/well) and the plate incubated further at 37 °C in an atmosphere of 5% CO 2 for 4 h. Finally, dimethyl sulfoxide was added (50 μL/well) to dissolve formazon crystals and the OD was measured using an enzyme-linked immunosorbent assay plate reader at 570 nm to determine the Stimulation Index (SI).

Statistical analyses.
Statistical analyses were carried out with JMP (www. jmp. com) using the analysis of variance (ANOVA) and Student's t-test. Data were analyzed using two-way repeat measures ANOVA followed by Bonferroni post-hoc test (when there were more than two time points) to detect differences between treatment groups. Differences were considered significant where P < 0.05. The correlation plot was prepared using SPSS 16.0. Bar charts were prepared using Prism 8.0 (GraphPad, San Diego, CA, USA).

Results
Characterization of chMoDCs. Cultured cells were characterized as chMoDCs based on their morphologic changes on alternate days and mRNA expression of CD14 and CD83 (maturation marker). Cell aggregation due to proliferation and stimulation of mononuclear cells by cytokines increased from day-2 to day-4 ( Fig. 1a-c). These aggregates sustained the growth and differentiation of mononuclear cells, exhibiting a veiled or dendritic appearance that was most pronounced on day-6 (Fig. 1d). When PBMCs were cultured under the similar conditions as above without adding GM-CSF and IL-4, cells exhibited no aggregation and dendritic appearance on respective days of culture. Following treatment with LPS or both Salmonella serovars, most cells exhibited a characteristic structure with extensive dendrites at 6 h post-treatment ( Fig. 1e-  www.nature.com/scientificreports/ gested that chMoDCs were in the final stages of maturation. Expression of CD14 mRNA on immature and LPStreated mature chMoDCs was compared with that obtained against unstimulated PBMCs. Immature chMoDCs showed relative downregulation (0.032 ± 0.0.002-fold), but CD14 expression was upregulated slightly in mature chMoDCs in comparison with that of unstimulated PBMCs (1.3 ± 0.02-fold; n = 3) (Fig. 2a). Besides, CD83 expression was upregulated on chMoDCs at 6 h (56-fold) and 24 h (16-fold) after LPS induction in comparison with that for immature phenotypes (P < 0.05) (Fig. 2b).

Intracellular bacterial survival in chMoDCs.
Gentamicin protection assay was employed to determine the intracellular bacterial survival in chMoDCs. The viable counts of S. Typhimurium recovered from chMoDCs was significantly higher than S. Gallinarum at 3 h post-infection (Fig. 3). Salmonella-infected chMoDCs showed a marginal increase in the viable counts of S. Gallinarum over a period from 3 to 24 h post-infection (P < 0.0001), however, S. Typhimurium remained almost constant or slightly decreased during this period (Fig. 3). This finding suggested that S. Gallinarum was multiplying in chMoDCs and was able to resist killing. At 48 h post-infection, the viable bacterial counts of both Salmonella serovars declined appreciably than that at 3 h post-infection, but S. Gallinarum continued to show higher persistence than that of S. Typhimurium (P < 0.0001). Hence, S. Gallinarum was able to resist killing by chMoDCs efficiently (Fig. 3 and Table 1).  Gallinarum group, which induced a high level of toxicity in chMoDCs at 3 h post-infection (P < 0.001) (Fig. 4). However, the level of cytotoxicity in the S. Gallinarum group was decreased at 48 h post-infection and was below the level of S. Typhimurium (P < 0.001) ( Fig. 4 and Table 1).  Table S2).    (Fig. 6b). mRNA expression of IL-1β was greatest among all other cytokines and chemokines at all time points in both groups, with the fold-change being greater in the S. Gallinarum group than that in the S. Typhimurium group (Fig. 6b).

TLR expression in response to
Neither Salmonella serovars showed a significant upregulation in IFN-γ mRNA expression as compared with that of mock-infected controls at any time point (Fig. 6b).  Table S4).

Stimulation of allogenic T-cells by Salmonella treated chMoDCs (MLR assay).
The function of chMoDCs exposed to S. Gallinarum or S. Typhimurium to stimulate proliferation of allogenic T-cells was tested and compared with that of mock-infected chMoDCs and between treatment groups. S. Typhimurium-exposed chMoDCs had a higher stimulatory potential towards allogenic T-cells (SI = 9.7) in comparison with that of S. Gallinarum infection (SI = 4.1) (P < 0.001) (Fig. 7).

Discussion
Mechanisms of host adaptation can differ between host-restricted and broad-host-range (host-generalist) strains of Salmonella serovars. Reports have suggested that Salmonella serovars differ in their ability to avoid adaptive immunity in mice because of interference with DC function, and that this interference feature of Salmonella is host-restricted 21 . To what extent S. Gallinarum modulates the function of chicken DCs (and whether this feature confers host specificity to this serovar) is not known. Therefore, we investigated in detail the interplay between host-restricted S. Gallinarum and host-generalist S. Typhimurium with chicken DCs. DCs derived from chicken bone marrow or blood monocytes have been cultured and characterized by several research groups 17,22,23 . We have used PBMCs as a source of monocytes and optimized the concentration of GM-CSF (25 ng/mL) and IL-4 (12.5 ng/mL) for the proper differentiation of monocytes into chMoDCs. In the present study, chMoDCs were grown and characterized according to their typical morphology and foldchange in mRNA expression of CD14 and CD83. Most cells exhibited a typical dendrite-like structure which www.nature.com/scientificreports/ was visible microscopically (Fig. 1d). In general, T-cells, DCs, and platelets are CD14-negative cells, but bone marrow-derived DCs can express CD14 to various extents during differentiation 24 . We observed a significant downregulation of CD14 mRNA expression in immature chMoDCs on day-6 of culture as compared with that of blood monocytes/PBMCs (which were originally taken on day-0 for in vitro culture of chMoDCs) (P < 0.0001). However, following treatment of chMoDCs with bacterial LPS at 24 h of incubation, we documented a slight increase in the fold-change expression of CD14 as compared with that of blood monocytes/PBMCs (1.3 ± 0.02fold) (Fig. 2a). This observation is in accordance with earlier report showing surface expression of CD14 to be upregulated positively with incubation of mouse bone marrow-derived DCs with LPS 25 . Therefore, CD14 could show marginal expression on mature chicken DCs. Reports describing CD14 expression on immature or mature phenotypes of chMoDcs have not yet been published. CD14 expression was observed on monocytes and mature chMoDCs, so it cannot be considered to be a specific marker for DC characterization. Another surface marker, CD83, which is invariably considered to be a maturation marker for DCs, has been reported in mammals and avian species 23,26 . The fold-change expression of CD83 on chMoDCs was upregulated significantly at 6 h and 24 h post-LPS treatment as compared with that in untreated cells (P < 0.05) (Fig. 2b). Besides LPS, both serovars of Salmonella favored maturation of chMoDCs in comparison with mock-infected cells. However, fold-change expression of CD83 mRNA (maturation level) was significantly greater in S. Typhimurium-infected chMoDC as compared with that in the S. Gallinarum group ( Fig. 5 and Supplementary Table S2). This finding suggested the potential of the host-restricted serovar S. Gallinarum for delaying the DC maturation. Both Salmonella serovars imparted pathologic/morphological changes in the chMoDCs in comparison with that in the mock-treated control. The chMoDCs treated with both the serovars got damaged as cells were found to be missing from within the cell aggregates with complete loss of dendrites which were evident at early stage of infection (6 h). These changes were more pronounced at 24 h post-infection (Fig. 1g). Use of a high ratio of Salmonella:DCs (10:1) might be the reason behind these pathologic changes because there have been reports of a reduction in the viability of murine DCs ≤ 50% upon infection with a higher number (10:1) of Salmonella per cell 27 . However, other scholars have reported no toxicity towards DCs even when 15 bacteria (S. Typhimurium) per cell were used 28 . A high ratio of bacteria:DCs is unlikely to equate to high ratios in vivo in the early stage of infection, but may be more relevant to the later stage of infection, when marked pathologic changes become apparent. Hence, a low-dose model of infection might be more relevant for studies on host-pathogen interactions because this may have a close resemblance to in vivo situations. This hypothesis warrants further studies using a low-dose model of infection.
The ability to survive in the intracellular environment is central to the pathogenesis of Salmonella infection. Our findings suggest that chMoDCs supported the replication of S. Gallinarum, which resulted in a marginal increase in their numbers over a period from 3 to 24 h post-infection (P < 0.0001), though the initial viable counts at 3 h post-infection was significantly higher for S. Typhimurium (Fig. 3 and Table 1). Recovery of high viable counts of S. Typhimurium at 3 h post-infection may be due to its relatively higher invasive potential and low cytotoxicity, in agreement with earlier publication 29 . The survival of S. Gallinarum at 24 h and 48 h postinfection was higher than that of S. Typhimurium (P < 0.0001), which suggested that, between the two serovars, S. Gallinarum was more resistant to intracellular killing by chMoDCs (Fig. 3 and Table 1). Our data show that Salmonella could persist in infected chMoDCs for ≥ 48 h post-infection, though the survival of bacteria declined over time. This could be one of the factors responsible for the higher persistence and disease-causing ability of www.nature.com/scientificreports/ S. Gallinarum in chickens as compared with that of S. Typhimurium because the survival for S. Gallinarum was notably higher in chMoDCs than that of S. Typhimurium. This finding is contrary to reports showing host-restricted and host-generalist Salmonella serovars do not exhibit a marked difference in their resistance to killing of chicken macrophages at 48 h of infection 15 . The reason for this discrepancy might be due to the use of different phagocytic cells (DCs vs. macrophages), which could modulate the replication of bacteria differently. Salmonella species have been shown to promote the death of host cells as early as 1 h post-infection in a SPI-2 T3SS-dependent manner 30,31 . S. Gallinarum-infected chMoDCs showed a significantly higher level of cytotoxicity (29.67 ± 9.11) at 3 h post-infection in comparison with that of S. Typhimurium (2.37 ± 4.03), which was relatively more cytotoxic at the late phase of infection (48 h) (Fig. 4 and Table 1). Our results are in accordance with a previous report which revealed that host-restricted Salmonella serovars such as S. Typhimurium induced rapid cytotoxicity in mouse bone marrow derived DCs at 3 h post-infection 32 . This could probably dampens the adaptive immunity against this gut pathogen and has been proposed as an early immune escape mechanism for subsequent systemic spread. Hence, we propose that the capacity of S. Gallinarum to restrict the activation of chMoDCs during early stage of infection may be linked with its higher cytotoxic potential at 3 h post-infection.
We measured cytotoxicity in the early phase (3 h) and late phase of infection (48 h). By doing so, it was possible to distinguish between strains that were recovered in low counts due to cytotoxicity and lysis of chMoDCs and those that succumbed to DC defenses. Hence, our cytotoxicity results were subjected to correlation with the intracellular bacterial survival assay which revealed no correlation between these assays for both the serovars over a period of time except for S. Gallinarum. The correlation plot revealed a significant positive correlation between these two assays at 48 h post-infection for S. Gallinarum (P < 0.05) (see Supplementary Fig. S1). A clear link to the superior survival of S. Gallinarum in chMoDCs was not evident by present data on intracellular survival assay because, after 48 h of infection, the level of cytotoxicity was very low for S. Gallinarum (5.77 ± 2.55%) as compared to S. Typhimurium (35.3 ± 4.10%) but bacterial recovery was only marginally higher for former than later serovar, in agreement with previous report 15 (Figs. 3 and 4, Table 1). However, data for reduced cytotoxicity by S. Gallinarum at 48 h post-infection may not be correct in true sense as the replacement of media at 3 h post-infection witnessed loss of accumulated LDH and consequently low cytotoxicity. Indeed, the cumulative percentage cytotoxicity seem to be almost equal for both the serovars at 48 h post-infection. Nevertheless, further detailed studies on the interaction of chicken DCs with Salmonella are warranted to elucidate undefined host-specific traits.
DC maturation involves a coordinated series of events. An important hallmark of DC maturation is increased surface expression of MHC molecules and costimulatory molecules 33 . Expression of costimulatory molecules and MHC molecules was upregulated following treatment of chMoDCs with Salmonella serovars, findings that are consistent with those in other reports 28,34 . . S. Typhimurium-infected chMoDCs showed significant upregulation in the expression of costimulatory (CD40, CD80, CD83 and CD86) and MHC molecules at 6 h post-infection in comparison to S. Gallinarum (Fig. 5 and Supplementary Table S2). This proves that later Salmonella serovar had retarded the maturation of DCs especially during the early phase of infection (6 h). This is the first report describing the differential expression of costimulatory molecules and MHC molecules on chicken DCs in response to host-restricted and host-generalist Salmonella serovars.
Salmonella possesses a range of protein and non-protein structures that function as pathogen-associated molecular patterns, which are recognized by pattern recognition receptors such as TLRs expressed on DCs. TLR4 and TLR21 (which is functionally equivalent to mammalian TLR9) recognize and bind with their respective ligands LPS and Unmethylated (or 5′-C-phosphate-G-3′) DNA sequences are considered to be key players in immunity against Salmonella infection. We recorded a significant upregulation in expression of TLR4 and TLR21 in S. Typhimurium-infected chMoDCs in comparison with that in the S. Gallinarum group during the early phase of infection (6 h) ( Fig. 6a and Supplementary Table S3). This optimal stimulation of the innate immune response might be a contributing factor in the early clearance of S. Typhimurium, and poor induction of TLR expression by S. Gallinarum (especially during the early phase of infection) may be beneficial in establishing systemic infection by avoiding the initial innate immune response. Our observations stated above are contrary to those in a recent report of significant downregulation of expression of TLR2, TLR4 and TLR5 in S. Typhimurium-and S. Dublin-but not S. Gallinarum-infected HD11 cells at 6 h post-infection 35 . We hypothesize that use of opsonized bacteria at a low MoI (5) and different phagocytic cells might be the reason for this discrepancy because opsonized bacteria may not replicate efficiently inside cells and thereby lead to low induction of TLRs.
One of the most striking characteristics of DCs is their ability to produce cytokines and chemokines, which have important roles in regulating host immune responses upon Salmonella infection 36 . Hence, we evaluated the ability of chMoDCs to induce the transcription of several cytokines and chemokines in response to infection by S. Gallinarum or S. Typhimurium.
We found upregulation in expression of the proinflammatory cytokines IL-1β, IL-6, TNF-α, and IL-12p35 as well as the chemokine CXCLi1 in chMoDCs treated with S. Typhimurium during the early phase of infection (6 h). However, mRNA expression of only IL-1β, IL-6 and CXCLi2 was upregulated in the early phase (6 h) whereas mRNA expression of TNF-α, IL-12p35 and CXCLi1 was upregulated in the late phase (24 h) of S. Gallinarum infection (Fig. 6b and Supplementary Table S4). TNF-α is a crucial proinflammatory cytokine required during the early phase of infection and also during a specific immune response. TNF-α expression was downregulated during the initial phase (6 h) of S. Gallinarum infection. Switching to increased expression of TNF-α, IL-12p35 and CXCLi1 by S. Gallinarum-infected chMoDCs from 6 to 24 h post-infection might have been due to increased bacterial survival and higher expression of costimulatory molecules, which may have increased expression of these molecules synergistically during the late phase of infection (24 h). We concluded that S. Typhimurium triggered a strong inflammatory response which may limit the spread of bacteria largely to the gut. S. Gallinarum induced an inflammatory response that was not as strong as that induced by S. Typhimurium, especially during the early phase of infection. Hence, containment of infection was poor, and this could culminate www.nature.com/scientificreports/ in a severe systemic disease called fowl typhoid. Studies have revealed dominant proinflammatory-cytokine and chemokine responses by primary human and chicken epithelial cells as well as chicken macrophages in response to host-generalist serovar such as S. Typhimurium in comparison to host-restricted serovars 35,37,38 . Our findings are distinct in that S. Gallinarum did not completely retard the expression of proinflammatory cytokines as observed in epithelial cells 38 . However, their quantum was relatively narrower (IL-1β, IL-6 and CXCLi2) in comparison to S. Typhimurium (IL-1β, IL-6, TNF-α, IL-12p35 and CXCLi1) infected chMoDCs especially during early phase of infection (6 h) 29,35 . Induction of only a limited number of proinflammatory mediators by S. Gallinarum during the early phase of infection may correlate with its higher cytotoxic potential and poor capacity for DCs activation as revealed by low expression of co-stimulatory and MHC class II mRNA levels. There may be other contributing factors governing the differences in host responses against these serovars, including the difference in the early immune response.
There is a growing body of evidence suggesting that DCs can shape the T-helper type 1 (Th1)-Th2 balance, and that the latter is influenced primarily by the type of microbial interactions and their outcome 39 . This balance is governed by differential production of IL-12 and IL-4 by DCs because IL-12 induces IFN-γ-producing Th1 cells whereas Th2 responses are primed by IL-4 40 . We recorded significant upregulation of expression of IL-12p35, IL-4 and IL-10 and a slight increase in IFN-γ expression by S. Typhimurium-infected chMoDCs in comparison with that in the S. Gallinarum group at the early phase of infection (6 h), whereas S. Gallinarum favored strong upregulation of mRNA expression of IL-4, IL-10 and IL-12p35 (marginal increase) at the late phase of infection (24 h) ( Fig. 6b and Supplementary Table S4). Hence, S. Typhimurium-treated DCs may favor polarization of naïve T-cells to Th1 cells and Th2 cells, which could facilitate early clearance from the host, whereas S. Gallinarum favored primarily the Th2 response during the late phase of infection. This diminished expression of Th1-polarizing cytokines by chMoDCs in response to S. Gallinarum points towards a suppressed immune response in the form of cell-mediated immunity against this intracellular pathogen. This action may be one of the ways by which S. Gallinarum modulates the defense of DCs, by interfering with their ability to stimulate naïve T-cells through production of Th1 cytokines and, thus, helping their survival within chMoDCs. This observation is consistent with a finding by Tang and colleagues indicating that, in comparison with a host-generalist Salmonella serovar (S. Enteritidis), a host-restricted serovar (S. Pullorum) failed to upregulate mRNA expression of IL-12 and IFN-γ in the spleen, which might have favored persistent infection in the spleen of infected chickens 41 .
It has been shown that Salmonella-exposed DCs favor the induction of allogeneic T-cell responses 42 . Coculture of Salmonella-infected chMoDCs with allogenic chicken T-cells led to a significant increase in proliferation of lymphocytes in comparison with the levels shown by allogenic chicken T-lymphocytes in the presence of mock-infected DCs (Fig. 7). S. Typhimurium-treated chMoDCs had a higher potential for allogenic T-cell stimulation than that of S. Gallinarum (P < 0.001). These observations point to an enhanced capacity of chMoDCs to degrade S. Typhimurium, which can lead to efficient presentation of bacteria-expressed antigens on MHC class-I and class-II molecules. Hence, the capacity of S. Gallinarum to interfere with DC function could prevent activation of T-cells against antigens derived from this pathogen, a finding that is in concert with an observation by Bruno et al. 43 . Consistent with these findings, the impaired capacity of S. Typhimurium to survive within chMoDCs for an extended period could explain (at least in part) why adaptive immunity in chickens could be activated against this host-generalist pathogen. This observation is consistent with a report showing that S. Typhi and S. Enteritidis (non-host adapted serovars for mice) were degraded by murine DCs as compared with S. Typhimurium (host-adapted serovar for mice) but this condition was reversed by using human DCs within which S. Typhi (host-adapted serovar for humans) could replicate and S. Typhimurium and S. Enteritidis (nonhost adapted serovars for humans) failed to replicate, and were degraded readily by these APCs 21 .
Taken together, our findings indicate that infection of chMoDCs with S. Gallinarum was characterized by low intracellular killing and delayed activation, possibly favoring long-term survival in the intracellular environment, and caused an overall low induction of proinflammatory responses along with a poor T-helper type-1 response. Overall, our results support a new component for the host specificity of S. Gallinarum: the capacity to interfere with DC function in chickens. This information could contribute to: (i) identification of new molecular factors determining the host specificity of S. Gallinarum; (ii) the design of new and improved vaccines against this intracellular pathogen.